The previous article covered the absorption and distribution of oligonucleotides (ONs). This one covers metabolism and excretion.
Metabolism of oligonucleotide (ONs) products differs little between species. Metabolites found only in humans, or at much higher proportions in humans, are rare. Endo- and exonucleases carry out the metabolism, and these enzymes show no species, sex, or age differences in mammals. That consistency does not make metabolism studies unnecessary. Metabolites left after cleavage of a few nucleotides can retain activity, so measuring the prototype ON alone underestimates what the molecule does. Smaller metabolites may also hybridize with off-target sequences and produce unintended toxic effects.
In Vitro Metabolism Stability and Metabolite Identification
In vitro metabolism is used during discovery to rank candidate stability. Metabolite types are usually similar across species even when the rates differ, so the choice of matrix decides whether the assay predicts in vivo stability. Plasma, target tissue cells, and homogenates are the usual choices; for local administration, use the local matrix.
Metabolite identification in vitro matters for small molecules. For ONs the value is limited, because species metabolism is so similar. Some companies file no metabolite identification data at the IND stage and still start clinical trials. Others skip in vitro work entirely and provide in vivo data to lower registration risk, and some support the decision with published evidence that the risk of omitting the study is low.
In Vivo Metabolism Studies
ONs metabolism pathways are predictable enough that OSWG and ICH guidelines ask for in vivo data before Phase III trials.
Unless PK/TK studies show a sex difference in prototype exposure, in vivo metabolism is usually examined in one sex. Plasma or urine are the main matrices. Plasma tracks tissue exposure more closely; urine adds the renal route. Tissue is an option when the organ is a principal delivery site, expresses the PD target, or retains the drug long term. Metabolite analysis in tissue is not a regulatory requirement, and whether to run it depends on the research question. LC-MS handles detection and can run alongside toxicology studies.
Metabolite Safety Testing
FDA guidance on metabolites sets a concern threshold at steady state: exposure above 10% of the prototype drug. The same threshold applies to ONs. New LNP components, peptides, or linkers that no approved drug has used also need a safety assessment.
Excretion Pathways and Study Design
OSWG and ICH M3 recommend excretion data before Phase III trials. Excretion routes are similar across ONs. Most of the dose is metabolized in tissue, and a small fraction is excreted as prototype drug.
Excretion can be studied in a dedicated PK study or alongside toxicology. Single-dose administration in one species is usually enough, and rodents are often used. Both prototype and metabolites are measured. A single dose at the intended clinical level normally suffices; if tissue distribution is part of the question, a higher dose may be needed. Urine is the default sample. When urine is not the main excretion route, collect bile instead, and add feces and breath if gastrointestinal or pulmonary toxicity appears.
Radiolabeling is not mandatory. Most marketed ONs used it to establish mass balance, but OSWG's position is that metabolism and excretion data for ONs are already sufficient and that radiolabeling similar products adds little. Radiolabeling also cannot run alongside a toxicology study. It still has uses: when other methods lack sensitivity, for new types of ONs products, and for tissues with retention long enough to make clinical radiolabeled excretion studies impractical. Where radiolabeling is used, place the label in a region stable to nucleases.
Breast milk secretion depends on exposure level and duration, oral bioavailability, and whether lactating women are in the target population. ICH S5(R3) generally does not require the study. Data from marketed ONs show very low or undetectable concentrations in milk.
As with absorption, distribution, and metabolism, any conjugate or formulation component that is not the ON itself has to be added to the excretion tests. mRNA therapeutics are the exception: they are inherently unstable, so excretion studies are not required.
In Vitro Drug Interaction Assessment
DDI work centers on metabolic enzymes and transporters. A drug can act as a victim, with its own PK altered, or as a perpetrator that changes the PK of other drugs. As a victim, an ON would have to be a substrate for Phase I or Phase II enzymes or for a transporter. ONs are metabolized by nucleases, not CYPs, and they generally do not displace small molecules from plasma protein binding, so substrate studies are usually unnecessary. Several exceptions are worth checking: conjugated ONs or LNP formulations with new components may warrant enzyme substrate studies, ONs cleared mainly by the kidney can be assessed as renal transporter substrates, and GalNAc conjugates raise the question of hepatic efflux transport.
As a perpetrator, an ON can bind large molecules nonspecifically. Affinity is weak, but binding can still occupy enzyme or transporter active sites and shift the PK of a co-administered drug. Structure is not the whole story; conjugate and formulation components carry risk of their own. Platform programs can lean on earlier DDI data: a positive result in a precursor product means later products from the same platform need the study, and a negative result allows other product data and literature to stand in. DDI assessment normally uses the full-length material and excludes metabolites. A small fraction of an ON escapes the lysosome into the cytoplasm where metabolic enzymes sit, and hepatocytes model that path better than hepatic microsomes, which produce more false positives. Transporters are a real finding: casimersen inhibits MATE1, and eteplirsen inhibits OCT1 and OATP1B1. Inhibition testing across efflux and influx transporters is worth running.
ONs can also change CYP or transporter expression through their PD effects, which is a second DDI route. Literature review should establish whether the intended PD effect touches metabolic enzymes or transporters. FDA pays attention to this indirect route: cytokines regulate CYP expression, and ONs can shift cytokine concentrations. Standard in vitro CYP methods do not capture that, so plasma from dosed patients applied to hepatocytes is one option.
FDA (2020) and EMA (2012) guidance asks for DDI studies on small molecules before Phase III, and the same timing applies to ONs.
ADME Strategy for Oligonucleotide Drugs
Oligonucleotide drugs are a recent therapeutic class with no dedicated ADME guidance. Industry groups such as OSWG have supplied detailed recommendations in place of one. Most design principles carry over from small molecules and biologics, with several class-specific differences. Sex differences in absorption are unlikely, so single-species PK studies suffice. Sampling schedules should follow tissue half-life rather than plasma half-life. Tissue distribution studies matter more than they do for small molecules and need to happen earlier. Because metabolic enzymes are consistent across species, metabolism studies carry less weight than they do for small molecules. ONs are generally not substrates for hepatic CYPs, but they can change CYP or transporter activity, so DDI risk needs attention. Not every study has to be complete before an IND; many can be delivered during clinical development. Conjugated and delivery-vehicle components introduce additional risk, and study design should account for them because they change what the assays detect.
